A paper copy of the Sequence Listing and a computer readable form of the sequence listing on diskette, containing the file named SeqList.txt, which is 103 kilobytes in size (measured in MS-DOS), and which was created on Apr. 11, 2002.
The present invention is directed to proteins, nucleic acid sequences and constructs, and methods related thereto.
Fatty acids are organic acids having a hydrocarbon chain of from about 4 to 24 carbons. Many different kinds of fatty acids are known which differ from each other in chain length, and in the presence, number and position of double bonds. In cells, fatty acids typically exist in covalently bound forms, the carboxyl portion being referred to as a fatty acyl group. The chain length and degree of saturation of these molecules is often depicted by the formula CX:Y, where xe2x80x9cXxe2x80x9d indicates number of carbons and xe2x80x9cYxe2x80x9d indicates number of double bonds.
The production of fatty acids in plants begins in the plastid with the reaction between acetyl-CoA and malonyl-ACP to produce acetoacetyl-ACP catalyzed by the enzyme, xcex2-ketoacyl-ACP synthase III. Elongation of acetyl-ACP to 16- and 18-carbon fatty acids involves the following cycle of reactions: condensation with a two-carbon unit from malonyl-ACP to form a xcex2-ketoacyl-ACP (xcex2-ketoacyl-ACP synthase), reduction of the keto-function to an alcohol (xcex2-ketoacyl-ACP reductase), dehydration to form an enoyl-ACP (xcex2-hydroxyacyl-ACP dehydrase), and finally reduction of the enoyl-ACP to form the elongated saturated acyl-ACP (enoyl-ACP reductase). xcex2-ketoacyl-ACP synthase I, catalyzes elongation up to palmitoyl-ACP (C16:0), whereas xcex2-ketoacyl-ACP synthase II catalyzes the final elongation to stearoyl-ACP (C18:0). The longest chain fatty acids produced by the FAS are typically 18 carbons long. Additional biochemical steps in the cell produce specific fatty acid constituents, for example through desaturation and elongation.
xcex2-ketoacyl synthases, condensing enzymes, comprise a structurally and functionally related family that play critical roles in the biosynthesis of a variety of natural products, including fatty acids, and the polyketide precursors leading to antibiotics, toxins, and other secondary metabolites. xcex2-ketoacyl synthases catalyze carbon-carbon bond forming reactions by condenisng a variety of acyl chain precursors with an elongating carbon source, usually malonyl or methyl malonyl moieties, that are covalently attached through a thioester linkage to an acyl carrier protein. Condensing enzymes can be part of multienzyme complexes, domains of large, multifunctional polypeptide chains as the mammalian fatty acid synthase, or single enzymes as the xcex2-ketoacyl synthases in plants and most bacteria.
Condensing enzymes have been identified with properties subject to exploitation in the areas of plant oil modification, polyketide engineering, and ultimately design anti-cancer and anti-tuberculosis agents. One of the molecular targets of isoniazid, which is widely used in the treatment of tuberculosis, is KAS. Cerulinin, a mycotoxin produced by the fungus Cephalosporium caerulens, acts as a potent inhibitor of KAS by covalent modification of the active cysteine thiol. Condensing enzymes from many other pathways and sources have all been shown to be inactivated by this antibiotic with the exception of the synthase from C. caerulens and KAS III, the isozyme responsible for the initial condensation of malonyl-ACP with acetyl-CoA in plant and bacterial fatty acid biosynthesis. Inhibition of the KAS domain of fatty acid synthase by cerulinin is selectively cytotoxic to certain cancer cells.
The present invention is directed to xcex2-ketoacyl ACP synthase (KAS), and in particular to engineered KAS polypeptides and polynucleotides encoding engineered KAS proteins having a modified substrate specificity with respect to the native (also referred to herein as wild-type) KAS protein. The engineered polypeptides and polynucleotides of the present invention include those derived from plant and bacterial sources.
In another aspect of the invention polynucleotide encoding engineered polypeptides, particularly, polynucleotides that encode a KAS protein with a modified substrate specificity with respect to the native KAS protein, are provided.
In a further aspect the invention relates to oligonucleotides derived from the engineered KAS proteins and oligonucleotides which include partial or complete engineered KAS encoding sequences.
It is also an aspect of the present invention to provide recombinant DNA constructs which can be used for transcription or transcription and translation (expression) of an engineered KAS protein having an altered substrate specificity with respect to the native KAS protein. In particular, constructs are provided which are capable of transcription or transcription and translation in host cells. Particularly preferred constructs are those capable of transcription or transcription and translation in plant cells.
In another aspect of the present invention, methods are provided for production of engineered KAS proteins having a modified substrate specificity with respect to the native KAS in a host cell or progeny thereof. In particular, host cells are transformed or transfected with a DNA construct which can be used for transcription or transcription and translation of an engineered KAS. The recombinant cells which contain engineered KAS are also part of the present invention.
In a further aspect, the present invention relates to methods of using the engineered polynucleotide and polypeptide sequences of the present invention to modify the fatty acid composition in a host cell, as well as to modify the composition and/or structure of triglyceride molecules, particularly in seed oil of oilseed crops. Plant cells having such a modified triglyceride content are also contemplated herein.
The modified plants, seeds and oils obtained by the expression of the plant engineered KAS proteins are also considered part of the invention.